A week ago Howard Shapiro enlightened me (and maybe others) by
saying that, whereas propidium is (supposedly) membrane impermeant to
healthy cells, ethidium is membrane permeant and is pumped out of
healthy cells. Thus, propidium is a viability stain that operates by
passive dye exclusion and ethidium requires active participation on the
part of the healthy cell to appear negative for staining with this dye.
I am wondering if newly thawed cells have the ability to pump out
ethidium -- or, more specifically, ethidium monoazide (EMA) -- if they
are otherwise viable at that point. When I thaw cryopreserved cells I
get a clear-cut idea of their viability immediately afterward by trypan
blue exclusion (I realize that early apoptotic cells are trypan
negative) because it is a yes-or-no decision. When I stain with 5 ug/mL
EMA, leave the cells in the dark for 15 minutes and then expose them to
a nearby fluorescent light source for 15 minutes, I get a confusing
pattern when the cells are analyzed by flow cytometry after washing,
fixing, etc. Not even all of the cells obviously dead by FSC, SSC, and
hypodiploidy are positive for EMA staining. And the seemingly viable
cells have a wide range of EMA positivity, whereby many of them have
higher intensity than the lower intensity dead cells.
So, can EMA even be used right after cells have been thawed, or is
their permeability/dye pumping status totally messed up? I would like to
be able to quickly fix/permeabilize cryopreserved cells after thawing to
prevent further cell death and in preparation for intracellular staining
but be able to distinguish the cells that were dead prior to that point.
Also, is EMA unstable in solution at 4 C or RT, unlike plain old
ethidium bromide? The results I got soon after making a stock solution
of 0.5 mg/mL EMA were quite different from those 2 weeks later when the
stock had been sitting in the refrigerator.
Kevin G. Waddick, Ph.D.
Parker Hughes Institute
2657 Patton Road
St. Paul, MN 55113
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